Micro-evolutionary analysis of 70 ST398 isolates by pulsed-field gel electrophoresis (PFGE) using Cfr9I revealed three sub-clones with abundant inter- and intra-sub-clone heterogeneity in spa- and SCC mec-types. In addition, we developed two specific PCRs for the detection of Staphylococcus aureus sequence type 398 (ST 398) isolates with 100% specificity and high sensitivity.